Bottom Line:
We found that MSCs underwent aging and spontaneous osteogenic differentiation upon regular culture expansion, with progressive downregulation of TERT and upregulation of osteogenic genes such as Runx2 and ALP.Meanwhile, the expression of genes associated with stem cell self-renewal such as Oct4 and Sox2 declined markedly.Our results indicate that histone H3 acetylation, which can be modulated by extrinsic signals, plays a key role in regulating MSC aging and differentiation.

Background: Mesenchymal stem cells (MSCs) hold great promise for the treatment of difficult diseases. As MSCs represent a rare cell population, ex vivo expansion of MSCs is indispensable to obtain sufficient amounts of cells for therapies and tissue engineering. However, spontaneous differentiation and aging of MSCs occur during expansion and the molecular mechanisms involved have been poorly understood.

Methodology/principal findings: Human MSCs in early and late passages were examined for their expression of genes involved in osteogenesis to determine their spontaneous differentiation towards osteoblasts in vitro, and of genes involved in self-renewal and proliferation for multipotent differentiation potential. In parallel, promoter DNA methylation and hostone H3 acetylation levels were determined. We found that MSCs underwent aging and spontaneous osteogenic differentiation upon regular culture expansion, with progressive downregulation of TERT and upregulation of osteogenic genes such as Runx2 and ALP. Meanwhile, the expression of genes associated with stem cell self-renewal such as Oct4 and Sox2 declined markedly. Notably, the altered expression of these genes were closely associated with epigenetic dysregulation of histone H3 acetylation in K9 and K14, but not with methylation of CpG islands in the promoter regions of most of these genes. bFGF promoted MSC proliferation and suppressed its spontaneous osteogenic differentiation, with corresponding changes in histone H3 acetylation in TERT, Oct4, Sox2, Runx2 and ALP genes.

Conclusions/significance: Our results indicate that histone H3 acetylation, which can be modulated by extrinsic signals, plays a key role in regulating MSC aging and differentiation.

Mentions:
Previous studies have shown that MSCs are genetically stable after limited expansion [21]. So we investigated whether MSCs underwent epigenetic changes during culture expansion, with an emphasis on promoter DNA methylation and histone H3 acetylation. We found dramatic alterations in histone H3 acetylation in K 9 and K14 in TERT, Sox2, Oct4, Runx2 and ALP genes in replicating MSCs when comparing passage 1 with passage 6 MSCs (Fig. 4B; Fig. 5A, 5D, 6A and 6D). Downregulation in the expression of TERT, Sox2 and Oct4 genes were closely associated with decreases in H3 acetylation levels in the promoter regions of the corresponding genes (Fig. 4B and 4C; Fig. 5A, B, D and E). Meanwhile, upregulated expression of Runx2 and ALP genes was accompanied by increases in H3 acetylation levels in the promoter of the corresponding genes (Fig. 6A, B, D and E). We also examined DNA methylation in CpG islands in the promoter and exon 1 regions of these genes in parallel. Our results showed alterations in methylation levels in most of these genes in MSCs undergoing culture expansion, but they did not correlate to expressional changes of the corresponding genes except for ALP (Fig. 4C and D; Fig. 5B, C, E and F; Fig. 6 B, C, E and F).

Mentions:
Previous studies have shown that MSCs are genetically stable after limited expansion [21]. So we investigated whether MSCs underwent epigenetic changes during culture expansion, with an emphasis on promoter DNA methylation and histone H3 acetylation. We found dramatic alterations in histone H3 acetylation in K 9 and K14 in TERT, Sox2, Oct4, Runx2 and ALP genes in replicating MSCs when comparing passage 1 with passage 6 MSCs (Fig. 4B; Fig. 5A, 5D, 6A and 6D). Downregulation in the expression of TERT, Sox2 and Oct4 genes were closely associated with decreases in H3 acetylation levels in the promoter regions of the corresponding genes (Fig. 4B and 4C; Fig. 5A, B, D and E). Meanwhile, upregulated expression of Runx2 and ALP genes was accompanied by increases in H3 acetylation levels in the promoter of the corresponding genes (Fig. 6A, B, D and E). We also examined DNA methylation in CpG islands in the promoter and exon 1 regions of these genes in parallel. Our results showed alterations in methylation levels in most of these genes in MSCs undergoing culture expansion, but they did not correlate to expressional changes of the corresponding genes except for ALP (Fig. 4C and D; Fig. 5B, C, E and F; Fig. 6 B, C, E and F).

Bottom Line:
We found that MSCs underwent aging and spontaneous osteogenic differentiation upon regular culture expansion, with progressive downregulation of TERT and upregulation of osteogenic genes such as Runx2 and ALP.Meanwhile, the expression of genes associated with stem cell self-renewal such as Oct4 and Sox2 declined markedly.Our results indicate that histone H3 acetylation, which can be modulated by extrinsic signals, plays a key role in regulating MSC aging and differentiation.

Background: Mesenchymal stem cells (MSCs) hold great promise for the treatment of difficult diseases. As MSCs represent a rare cell population, ex vivo expansion of MSCs is indispensable to obtain sufficient amounts of cells for therapies and tissue engineering. However, spontaneous differentiation and aging of MSCs occur during expansion and the molecular mechanisms involved have been poorly understood.

Methodology/principal findings: Human MSCs in early and late passages were examined for their expression of genes involved in osteogenesis to determine their spontaneous differentiation towards osteoblasts in vitro, and of genes involved in self-renewal and proliferation for multipotent differentiation potential. In parallel, promoter DNA methylation and hostone H3 acetylation levels were determined. We found that MSCs underwent aging and spontaneous osteogenic differentiation upon regular culture expansion, with progressive downregulation of TERT and upregulation of osteogenic genes such as Runx2 and ALP. Meanwhile, the expression of genes associated with stem cell self-renewal such as Oct4 and Sox2 declined markedly. Notably, the altered expression of these genes were closely associated with epigenetic dysregulation of histone H3 acetylation in K9 and K14, but not with methylation of CpG islands in the promoter regions of most of these genes. bFGF promoted MSC proliferation and suppressed its spontaneous osteogenic differentiation, with corresponding changes in histone H3 acetylation in TERT, Oct4, Sox2, Runx2 and ALP genes.

Conclusions/significance: Our results indicate that histone H3 acetylation, which can be modulated by extrinsic signals, plays a key role in regulating MSC aging and differentiation.